A member of Flaviviridae, the flavivirus family, the dengue virus (DENV) arguably causes more pain, suffering, and economic hardship than any other mosquito-borne viral pathogen. Currently, there is no effective, specific treatment for infection, and methods proposed for controlling dengue virus by vector eradication (WHO, 2014) and vaccination (Dengue Vaccine Initiative, 2015) have been largely ineffectual, though such efforts continue. Other pathogenic arboviruses among the Flaviviridae cause Zika virus infection, yellow fever, West Nile virus disease, tick-borne encephalitis, Japanese encephalitis, and other insect-borne infectious diseases.
According to the World Health Organization (WHO), dengue fever incidences have increased dramatically in terms of number, severity of cases, and geographic scope over recent decades. Presently, WHO estimates that 40 percent of the world's population (˜2.5 billion human beings) is at risk from dengue, that as many as 100 million new infections will occur each year and, that of these, 500,000 individuals should be hospitalized; unfortunately, most of those now afflicted have no access to adequate medical intervention. The dengue virus is carried by the mosquito, Aedes aegypti (and several less prominent species), and the range of this vector has been observed to have moved north and west in recent decades. In fact, in 2013, a number of cases were documented as having been contracted in Yunnan Provence, China and in Florida, US. Dengue infections present as flu-like illnesses that (increasingly, in recent years) progress to a more severe form known as Dengue Haemorrhagic Fever (aka, “severe dengue”) that, in clinical settings, recently has been conflated with Ebola virus disease. Dengue is endemic in tropical and subtropical regions, worldwide, and is becoming increasingly problematic in urban and semi-urban areas of most Asian, Latin American, and Western Pacific countries. Severe dengue is a leading cause of hospitalization and death among children in affected regions. The virus occurs as four distinct, closely-related serotypes: DENV1, -2, -3, & -4. Again, like Ebola, although there is no specific treatment for dengue fever, early detection and proper medical attention can lower fatality rates to less than one percent. Unfortunately, although eventual resolution of episodic dengue infection seems to confer lifelong immunity against the offending clade, the patient remains largely susceptible to the other clades and, with successive infections, the chances of developing severe dengue increase significantly (WHO, 2014).
Like dengue, Zika is carried by mosquitoes of genus Aedes and, currently, there is no preventative or therapeutic directly active against the virus. Historically (since its original isolation in 1947), Zika infections occur asymptomatically in 80% of those affected and disease symptoms are common cold-like, and self limiting. Recently, Zika virus infections have been documented as showing significant increases in incidence, severity, and geographic scope. Owing to dramatic outbreaks in South America beginning in 2015; having moved beyond Tropical Africa and Southeast Asia; and its apparent capacity for teratogenicity causative of microcephaly and other CNS developmental problems; the fact that Zika may also be transmitted via sexual contact; and increased, associated occurrences of Guillain-Barre' syndrome, Zika has been classified as an “emerging disease” by The European Centre for Disease Prevention and Control (ECDC, 2016).
Enveloped viruses, once injected into a human host, are moved by thermodynamic diffusion until close enough to potential host cells for complementary membrane receptors to mediate adsorption and attachment of virions to cells. Flavivirus structural E protein receptors mediate both binding with and fusion to animal cells and are labeled as “class II” viral fusion proteins. The term “fusion machines” was coined to describe the dynamics of proficient cell ingress engineered by, so far as is now known, classes I through III viruses. Although the structural characteristics between the three classes are quite different, the mission supported is always the same—cell membrane recognition, attachment, and lipid bilayer fusion to accomplish cell entry by voracious, predatory viruses (Kielian, 2006; White, et al., 2008).
Among flaviviruses and alphaviruses, class II proteins are seen evolutionarily to have developed such that, although structurally diverse, they are mechanistically related in terms of their fusion-enabling architecture. As seen in class I proteins, a proteolytic cleavage occurs in class II virions yielding mature virions bearing primed fusion proteins. In the case of alphaviruses, pE2 proteins are cleaved to E2 and, for flaviviruses, the corresponding proteins are PrM and M. Again, as for class I viruses, the critical conformational changes activating fusion in class II are initiated by exposure to low pH that, in this case, reveals the fusion peptide formerly protected as a buried internal loop at the end of an elongated subdomain (Strauss & Strauss, 1994).
Although some of the pre-fusion structures of flaviviral and alphaviral envelope proteins have been determined, much work remains to be done in this area of research and the exact mechanism underlying the modus operandi of class II viral fusion proteins remains to be elucidated. For this reason, at least in part, no therapeutic specifically able to inhibit the fusion of such proteins and consequent infectivity of this virus class has been developed (van der Schaar, et al., 2007). An urgent, unmet need currently exists for the discovery and practical development of cell-entry inhibitors specifically designed to inhibit infection by flaviviruses, alphaviruses, and hepatitis viruses. Given, membrane fusion is a vital step in the progression of class II viral pathogenesis, a more complete description of the structure and function of envelope proteins is required for purposes of producing effective therapeutic and preventative agents against flavi-, alpha-, and hepatitis viruses.
U.S. Pat. No. 8,541,377 (the '377 patent) discloses peptides that are virus-to-cell fusion- and entry-inhibitors bindable to regions in class II E viral proteins. That patent discloses compounds and methods for screening compounds potentially active against these bindable regions to discover therapeutic candidates for fighting disease viruses bearing class II E proteins; these include dengue fever, dengue hemorrhagic fever, tick-borne encephalitis, West Nile virus disease, yellow fever and, possibly, hepatitis C. Further, it discloses a method for identifying E protein topographical regions bindable by an artificially-synthesizable and highly specific peptide. Said peptide can be configured demonstrably to act with sufficient degrees of affinity and avidity; further, such virus binding can be assayed either in vitro and, likely, in vivo. Further, it also may be utilized in structure determination, drug screening, drug design, and other methods described and claimed in the '377 patent. Methods for inhibiting viral infection, measuring same, and (theoretically) treating dengue-induced diseases are provided using methods that employ peptides and derivatives thereof to inhibit dengue virus-to-cell binding using a cell entry-inhibitory peptide comprised of 28 amino acids (AAs) arranged in the following sequence: (seq. intentionally redacted), identified as DN81 and labeled SEQ ID NO: 1 for purposes of and within, that particular patent.
No particular isomeric form of DN81 is disclosed within the '377 patent. This, ordinarily, is not surprising because when a given peptide sequence is found to have a desired property—for example, binding to a particular receptor site—it is expected that one isomeric form, and only one isomeric form of the peptide—within a racemic mixture—will have an effective binding conformation. The other isomeric form, e.g., the L- or D-enantiomer, will have no, or very little, binding capability. The obvious reason for effective binding being provided by only one isomeric form is that such binding is highly related to, and controlled by, the 3-dimensional conformation of the peptide and its ability to position a residue sequence aligning with a residue sequence forming a binding site(s) within or upon a target virion. Thus, binding between such peptides and a target AA sequence found, for example, within the flavivirus II E envelope protein, is controlled by the 3-dimensional conformation of both binding site and a putative therapeutic peptide under physiological conditions. Thus, considering L- and D-forms of such binding peptides display “mirror image” conformations, it is expected and ordinarily found that only one isomer (virtually always, L-) is biologically active and can participate in effective binding.
Despite the fact that the binding qualities of a therapeutic peptide are provided by only one enantiomeric form, knowing which particular isomeric form is effective is unnecessary as, ordinarily, there will be a sufficient concentration of the effective isomer within the racemic mixture to provide the desired effect.
Although not discussed in the '377 patent, DN81 (as were many different candidate peptides) was designed and synthesized exclusively as the enantiopure, L-form peptide for testing—as the “L-” enantiomer is almost always, as discussed above, the form effective in providing binding. Although oral dosing with DN81 was originally contemplated, among various, potentially less efficacious forms of administration for therapeutic purposes, it is now understood that digestion by peptidases effectively precludes this possibility. This is, of course, unfortunate as providing a therapeutic capable of oral administration would be expected to increase greatly the number of individuals who could and would accept treatment and, thereby, effectively reduce the number of individuals infected with, and harboring (and potentially disseminating), such viral pathogens.
Many areas of the world heavily affected by flaviviral infections also contain populations with severely limited access to modern medical technologies. Effectively treating, curing, and preventing such infections must be accomplished by means of a methodology that not only blocks viral entry, but also enables and encourages compliance through efficient administration of the therapeutic agent. Oral, as opposed to parenteral, administration would be highly favored for such purposes and, particularly so, for treating pediatric patients. Oral administration in medically-underserved regions, also should obviate the use of injections and the clear possibility of passing blood-borne infections such as HIV and viral hepatitides when needles and syringes are reused. Clearly, there is an absolute, heretofore, unmet need for the development of effective anti-flaviviral therapeutics that can be administered orally.
To develop a therapeutic peptide capable of binding with ligands within flaviviral class II E envelope proteins to interfere with virus-to-target cell binding and fusion would be highly desirable—and even more so—if such peptide were resistant to the degradative actions of peptidases. The development of a peptidase-resistant peptide would likely enable oral (and several other routes of administration that risk exposure to peptidases) delivery of said therapeutic to animals subject to flaviviral infections.